Figure 1 - uploaded by Concetta Gardi
Content may be subject to copyright.
( a ) Autofluorescence of freshly isolated HSC from rat liver. Cells examined under ultraviolet illumination at 328 nm revealed yellow fluorescence indicating the presence of vitamin A; expression of a -SMA. ( b ) Control: after 1 day, cells appear quiescent and negative for the marker; ( c ) by 7 days in culture, all the cells are activated. (original magnification  120).
Source publication
Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological...
Contexts in source publication
Context 1
... were harvested from the top of gradient and seeded (10 6 cells/ml) in 25 cm 2 flasks in Dulbecco's mod- ified Eagle's medium (DMEM) plus 20% fetal bovine serum and antibiotics. The viability and purity of preparation was tested by trypan blue exclusion and autofluorescence of vitamin A (Figure 1a), respec- tively. Cells were incubated in 95% air-5% CO 2 at 371C and allowed to adhere. ...
Context 2
... were incubated in 95% air-5% CO 2 at 371C and allowed to adhere. After 1 day, the cells appeared quiescent and negative for the marker a- SMA (Figure 1b), detected by means of Smooth muscle-alpha actin Immunoistology Kit (IMMH-2, Sigma, St Louis, USA). Experiments were performed at the second serial passage when the cells exhibited the a-SMA marker. ...
Similar publications
In vitro and in vivo studies indicate that oxidant stress is implicated in liver fibrogenesis. However, it is still unknown whether, in vivo, oxidant stress directly affects the hepatic cells responsible for fibrogenesis, ie, the hepatic stellate cells (HSCs). This study was aimed at answering this question by assessing the temporal and spatial rel...
Circulating endothelin-1 (ET-1) levels are increased in cirrhosis. The liver is an important site for circulating ET-1 clearance through the ET(B) receptor. We evaluated ET-1 kinetics in cirrhosis to determine if a reduced liver clearance contributes to this process. Cirrhosis was induced by carbon tetrachloride in rats. Hepatic ET-1 clearance was...
Anti-fibrotic and organ protective effects of brain natriuretic peptide (BNP) have been reported. In this study, effects of BNP on liver fibrosis were examined in the carbon tetrachloride (CCl(4))-induced liver fibrosis model using BNP-transgenic (Tg) and wild-type (WT) mice. Twice-a-week intraperitoneal injections of CCl(4) for 8 weeks resulted in...
Hepatic stellate cells (HSCs) express alpha-smooth muscle actin (alphaSMA) and acquire a profibrogenic phenotype upon activation by noxious stimuli. Insulin-like growth I (IGF-I) has been shown to stimulate HSCs proliferation in vitro, but it has been reported to reduce liver damage and fibrogenesis when given to cirrhotic rats.
The authors used tr...
Imatinib mesylate (STI-571), a tyrosine kinase inhibitor, has previously been demonstrated to attenuate liver fibrogenesis through inhibition of the activation of hepatic stellate cells (HSCs) in CCL(4)-treated rat models.
This study aimed to further evaluate the role of STI-571 in liver regeneration.
All animals were divided into four groups, and...
Citations
... Isoprostanes are considered as the most reliable markers of oxidative stress in a number of human pathologies [35]. In addition to being markers of oxidative stress, isoprostanes appeared to be the mediators of important biological effects such as stimulation of collagen synthesis [36], another hallmark of DD [5]. ...
Diverticular disease (DD) management is impaired by its pathogenesis, which is still not completely defined, with an unmet clinical need for improved therapies. Ex vivo DD human models demonstrated the presence of a transmural oxidative imbalance that supports an ischemic pathogenesis. This study aimed to assess, with the use of circulating biomarkers, insights into DD pathogenesis and possible therapeutic targets. Nox2-derived peptide, H2O2, antioxidant capacity, isoprostanes, thromboxanes, TNF-α, LPS and zonulin were evaluated by ELISA in healthy subjects (HS) and asymptomatic and symptomatic DD patients. Compared to HS, DD patients presented low antioxidant capacity and increase in sNox2-dp, H2O2 and isoprostanes paralleled to a TNFα increase, lower than that of oxidative markers. TxB2 production correlated to Nox2 and isoprostanes, suggesting platelet activation. An increase in zonulin and LPS highlighted the role of gut permeability and LPS translocation in DD pathogenesis. The increase of all the markers statistically correlated with DD severity. The present study confirmed the presence of a main oxidative imbalance in DD and provides evidence of platelet activation driven by LPS translocation. The use of circulating biomarkers could represent a new clinical tool for monitoring disease progression and validate therapeutic strategies never tested in DD as antioxidant supplementation.
... The exposure of rodent and human HSC/MFs to ROS or other redox-dependent reactive intermediates results in increased expression of ECM components. An initial study, performed on human HSC/MFs exposed to very low levels of HNE or to conditions leading to lipid peroxidation, showed a strong up-regulation of pro-collagen type I [94], a finding confirmed by subsequent studies employing other 4-hydroxy-2,3-alkenals of different chain lengths [95], as well as the other end-products of lipid peroxidation such as MDA [96] or F 2 -isoprostanes [97]. Along these lines, the same results were obtained when rat or human HSC/MFs were exposed to extracellularly available ROS such as H 2 O 2 or O 2 •− released by activated neutrophils, generated by the xanthine/xanthine-oxidase system, or by exposing HSC/MFs to the conditioned medium of normal hepatocytes undergoing oxidative stress [98][99][100]. ...
During chronic liver disease (CLD) progression, hepatic myofibroblasts (MFs) represent a unique cellular phenotype that plays a critical role in driving liver fibrogenesis and then fibrosis. Although they could originate from different cell types, MFs exhibit a rather common pattern of pro-fibrogenic phenotypic responses, which are mostly elicited or sustained both by oxidative stress and reactive oxygen species (ROS) and several mediators (including growth factors, cytokines, chemokines, and others) that often operate through the up-regulation of the intracellular generation of ROS. In the present review, we will offer an overview of the role of MFs in the fibrogenic progression of CLD from different etiologies by focusing our attention on the direct or indirect role of ROS and, more generally, oxidative stress in regulating MF-related phenotypic responses. Moreover, this review has the purpose of illustrating the real complexity of the ROS modulation during CLD progression. The reader will have to keep in mind that a number of issues are able to affect the behavior of the cells involved: a) the different concentrations of reactive species, b) the intrinsic state of the target cells, as well as c) the presence of different growth factors, cytokines, and other mediators in the extracellular microenvironment or of other cellular sources of ROS.
... 13 F2-IsoPs have been widely used as a biomarker of oxidative stress, prior studies have demonstrated the F2-IsoP's may also play direct signaling roles, including activation of hepatic stellate cells through the thromboxane-prostanoid receptor (TBXA2R). [14][15][16] In these studies, we determined that TBXA2R expression is increased during lung brosis, particularly in broblasts, where ligand binding by F2-IsoPs mediates broblast activation. Together, these investigations de ne a novel mechanism by which ROS contribute to brogenesis and implicate TBXA2R as a regulator of transforming-growth-factor beta (TGF-β) signaling. ...
While transient fibroblast activation is a normal and adaptive aspect of injury-repair in many contexts, persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF) and other chronic fibrotic lung diseases. The mechanisms regulating persistent fibroblast activation in IPF have not been fully elucidated. In the lungs of IPF patients and in mice with experimental lung fibrosis, we observed that expression of the thromboxane-prostanoid receptor (TBXA2R) was increased in fibroblasts. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, suggesting an alternative ligand activates profibrotic TBXA2R signaling. We found that F2-isoprostanes (F2-IsoPs), nonezymatic products of arachidonic acid metabolism generated in the setting of reactive oxygen species (ROS), are persistently elevated in experimental lung fibrosis and act as an alternative TBXA2R ligand. Further studies demonstrated that F2-isoprostanes signal through TBXA2R to activate Smad signaling, revealing TBXA2R as a previously unrecognized regulator of the transforming growth factor beta (TGF-β) pathway. Further, treatment with the small-molecule TBXA2R antagonist ifetroban protected mice from lung fibrosis in three pre-clinical models: bleomycin treatment, Hermansky-Pudlak Syndrome, and radiation-induced fibrosis. Importantly, treatment with ifetroban during the fibrotic phase of bleomycin injury markedly enhanced fibrotic resolution. Together, these studies implicate TBXA2R signaling as a crucial mediator linking oxidative stress to fibroblast activation and indicate that TBXA2R antagonists could be efficacious for treatment of IPF and other chronic fibrotic disorders.
... Besides being considered the most accurate and popular marker of lipid peroxidation, 24 F 2 -isoprostanes play a role in several pathological process including liver fibrogenesis. In a rat model of carbon tetrachloride-induced hepatic fibrosisHSCs) with F 2 -isoprostanes stimulates their activation to myofibroblasts25 and these effects are mediated by activation of receptors analogous to those for thromboxane A2 expressed on HSCs. 26,-isoprostanes show also potent vascular effects including vasoconstriction, monocyte adhesion to the endothelium, platelet aggregation and smooth muscular cells proliferation, 17 indicating that they are crucially involved in the atherosclerosis process. ...
Background and aims
HCV eradication improves non‐hepatic outcomes such as cardiovascular diseases although without clearly defined mechanisms. In this study we aimed to assess whether improvement of carotid atherosclerosis may be linked to a reduction of systemic oxidative stress after viral clearance.
Methods
We studied a retrospective cohort of 105 patients (age 62.4±11.2 years; 62 males) with F3/F4 fibrosis, characterized by carotid ultrasonography at baseline and at sustained virologic response (SVR) follow‐up. Levels of 8‐iso‐prostaglandin F2α (F2‐isoprostanes) and other oxidative stress markers were measured on frozen sera. Association between change (denoted as Δ) of oxidative stress markers (exposures) and change of carotid intima‐media thickness (cIMT) (outcome) was examined using multiple linear regression.
Results
Subclinical atherosclerosis, defined as the presence of carotid plaque and/or cIMT≥0.9, was present in 72% of the cohort. All patients achieved SVR that led to reduction of cIMT (0.92±0.20 vs 0.83±0.21 mm, P<0.001). HCV eradication markedly decreased serum levels of F2‐isoprostanes (620.5 [143.2; 1904.1] vs 119.51 [63.2; 400.6] pg/ml, P<0.0001), lipid hydroperoxides (13.8 [6.3; 20.7] vs 4.9 [2.3; 9.6] nmol/μl, P<0.0001) and 8‐hydroxy‐2'‐deoxyguanosine (558.9 [321.0; 6301.2] vs 294.51 [215.31; 408.95] pg/ml, P<0.0001) whereas increased serum GPx activity (10.44 [4.6; 16.3] vs 13.75 [9.42; 20.63] nmol/min/ml, P=0.001). By multiple linear regression analysis ΔcIMT was independently associated with ΔF2‐isoprostanes (β: 1.746 [0.948; 2.543]; P<0.0001) after adjustment for age, baseline F2‐isoprostanes and baseline IMT.
Conclusions
Besides association of lipid peroxidation with severity of liver disease, the reduction of F2‐isoprostanes may be involved in the improvement of atherosclerosis after HCV eradication.
... [19][20][21] 8-epi-PGF 2a induced hepatic stellate cell (HSC) proliferation and collagen production mediated liver fibrosis. [22][23][24] In addition, a recent study showed that the effect of 8-epi-PGF 2a on HSCs is dependent on the expression of the prostaglandin-related receptor TxA 2 r. [23,25] Therefore, we hypothesized that 8-epi-PGF 2a might promote PSC activation mediated through TxA 2 r. The purpose of this study was to investigate the role of TxA 2 r in the activation of PSCs induced by 8-epi-PGF 2a . ...
... [19][20][21] 8-epi-PGF 2a induced hepatic stellate cell (HSC) proliferation and collagen production mediated liver fibrosis. [22][23][24] In addition, a recent study showed that the effect of 8-epi-PGF 2a on HSCs is dependent on the expression of the prostaglandin-related receptor TxA 2 r. [23,25] Therefore, we hypothesized that 8-epi-PGF 2a might promote PSC activation mediated through TxA 2 r. The purpose of this study was to investigate the role of TxA 2 r in the activation of PSCs induced by 8-epi-PGF 2a . ...
Background. Pancreatic stellate cells (PSCs) activation plays a critical role in the development of chronic pancreatitis. Previous studies confirmed that thromboxane A2 receptor (TxA2r) was overexpressed in activated PSCs in rats. The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α (8-epi-PGF2α).
Methods. TxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay. Isolated PSCs were treated with 8-epi-PGF2α (10−6, 10−7, 10−8 mol/L) for 48 h, and SQ29548 (10−4, 10−6, and 10−7 mol/L), a TxA2r-specific antagonist for 48 h, respectively, to identify the drug concentration with the best biological effect and the least cytotoxicity. Then isolated PSCs were treated with SQ29548 (10−4 mol/L) for 2 h, followed by 10−7 mol/L 8-epi-PGF2α for 48 h. Real-time polymerase chain reaction was performed to detect the messenger RNA (mRNA) levels of α-smooth muscle actin (α-SMA) and collagen I. Comparisons between the groups were performed using Student's t test.
Results. TxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs (all P
... Besides being considered the most accurate and popular marker of lipid peroxidation, 24 F 2 -isoprostanes play a role in several pathological process including liver fibrogenesis. In a rat model of carbon tetrachloride-induced hepatic fibrosisHSCs) with F 2 -isoprostanes stimulates their activation to myofibroblasts25 and these effects are mediated by activation of receptors analogous to those for thromboxane A2 expressed on HSCs. 26,-isoprostanes show also potent vascular effects including vasoconstriction, monocyte adhesion to the endothelium, platelet aggregation and smooth muscular cells proliferation, 17 indicating that they are crucially involved in the atherosclerosis process. ...
... We previously demonstrated, in an experimental model of hepatic fibrosis, that F 2 -IsoPs are both markers of OS and mediators of fibrotic effects. The addition of F 2 -IsoPs to cultured hepatic stellate cells (HSC) greatly stimulated cell proliferation and collagen synthesis, two hallmarks of fibrosis [35]. In the liver, these fibrogenic events appear to be mainly mediated through the activation of thromboxane A2 (TxA 2r or TP receptor) receptors analogous [36,37]. ...
... With the aim to clarify the possible role of F 2 -IsoPs as mediators of fibrotic events, RLF were directly treated with 15-F 2t -IsoP. This resulted in a significant increase of cell proliferation and collagen synthesis, further confirming our previous data obtained in hepatic stellate cells [35,73,74]. ...
F2-isoprostanes (F2-IsoPs) have been considered markers of oxidative stress in various pulmonary diseases, but little is known about their possible role in pulmonary fibrosis. In this study, we have investigated the potential key role of F2-IsoPs as markers and mediators of bleomycin (BLM)-induced pulmonary fibrosis in rats. During the in vivo study, plasma F2-IsoPs showed a peak at 7 days and remained elevated for the entire experimental period. Lung F2-IsoP content nearly tripled 7 days following the intratracheal instillation of BLM, and by 28 days, the value increased about fivefold compared to the controls. Collagen deposition correlated with F2-IsoP content in the lung. Furthermore, from day 21 onwards, lung sections from BLM-treated animals showed α-smooth muscle actin (α-SMA) positive cells, which were mostly evident at 28 days. In vitro studies performed in rat lung fibroblasts (RLF) demonstrated that either BLM or F2-IsoPs stimulated both cell proliferation and collagen synthesis. Moreover, RLF treated with F2-IsoPs showed a significant increase of α-SMA expression compared to control, indicating that F2-IsoPs can readily activate fibroblasts to myofibroblasts. Our data demonstrated that F2-IsoPs can be mediators of key events for the onset and development of lung fibrosis, such as cell proliferation, collagen synthesis and fibroblast activation. Immunocytochemistry analysis, inhibition and binding studies demonstrated the presence of the thromboxane A2 receptor (TP receptor) on lung fibroblasts and suggested that the observed effects may be elicited through the binding to this receptor. Our data added a new perspective on the role of F2-IsoPs in lung fibrosis by providing evidence of a profibrotic role for these mediators in the pathogenesis of pulmonary fibrosis.
... An established trigger for HSC activation is oxidative stress, and, in particular, the products of lipid oxidation or oxidative modification of membrane polyunsaturated fatty acids. This class of molecules includes the reactive lipid aldehydes malondialdehyde, 4-hydroxy-2-nonenal (4-HNE), and F 2 isoprostanes (such as 8-iso-PGF2α) [4,5]. Importantly, exposure of HSC to 4-HNE leads to increased synthesis and deposition of collagen I [6,7], increased expression of matrix remodeling proteins, such as TIMP1 [7], activation of JNK signaling pathway [8], and increased pro-inflammatory signaling (MCP-1) [9]. ...
Aims:
Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E2-IsoLG and used it to investigate whether IsoLG could induce activation of HSC.
Results:
Primary human HSC were exposed to 15-E2-IsoLG for up to 48hours. Exposure to 5 μM 15-E2-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500 nM) 15-E2-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response.
Innovation:
This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis.
Conclusions:
IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.
... hepatic inflammation (Fig. 2). Also, lipid peroxidation of F 2 -isoprostanes in hepatocytes mediated activation of stellate cells, thereby leading to hepatic fibrosis [77]. Indeed, hepatic stellate cells (HSCs) have been established as the central hepatic cell in inducing a fibrotic response in the liver [78]. ...
Non-alcoholic steatohepatitis (NASH) is viewed as the hepatic manifestation of the metabolic syndrome and is a condition hallmarked by lipid accumulation in the liver (steatosis) along with inflammation (hepatitis). Currently, the etiology and mechanisms leading to obesity-induced liver inflammation are not clear and, as a consequence, strategies to diagnose or treat NASH in an accurate manner do not exist. In the current review, we put forward the concept of oxidized lipids as a significant risk factor for NASH. We will focus on the contribution of the different types of oxidized lipids as part of the oxidized low-density lipoprotein (oxLDL) to the hepatic inflammatory response. Furthermore, we will elaborate on the underlying mechanisms linking oxLDL to inflammatory responses in the liver and on how these cascades can be used as therapeutic targets to combat NASH. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder.
... Hepatocytes of plasma membrane contain Ca 2+ channels which admit Ca 2+ in response to receptor-operated channel agonists. The receptor-operated Ca 2+ channels allow various divalent cations to enter the hepatocyte [42]. Verapamil inhibit Ca 2+ entry into liver cells through its effect on receptor-operated channels [43]. ...
Fabrication and Characterization of ALK1fc-Loaded Fluoro-Magnetic Nanoparticles Rods for Inhibiting TGF β1 in HCC.
Nemany A.Hanafy1, 3, Marzia Maria Ferraro1, Antonio Gaballo1, Luciana Dini2, Vittorianna Tasco1, Concetta Nobile1, Maria Luisa De Giorgi3, Sonia Carallo1, Ross Rinaldi3 and Stefano Leporatti1
1 CNR NANOTEC- Istituto di Nanotecnologia Lecce (Italy).
2 University of Salento, DISTEBA, Lecce (Italy).
3 University of Salento, Mathematics and Physics Department, Lecce (Italy).
Email: nemany.hanafy@nanotec.cnr.it
Abstract
Fluoro-magnetic nanoparticles (f-MNPs) are needed strongly as contrast agent for bio-medical- fluorescence imaging [1]. Indeed, their size and concentration in the tumor allow a very high resolution and an accurate mapping of lesions. Fluorescence isothiocyanate that was added during fabrication, has been entrapped inside crystals of MNPs during crystallization. This led to a change of crystal architecture giving rise to elongated rods. Ferric atoms were distributed over the length of crystal leading to strengthening of magnetic intensity [2]. TEM and SEM-EDX show that elongated crystals were produced with high ferric atoms scattering. The intensity of fluoro-MNPs fluorescence was detected by fluorescence spectrophotometer and confocal microscope. The benzene ring structure of FITC and its carboxylic group were clearly detected by using FTIR, compared to MNPs prepared in absence of FITC. F-MNPs rods were functionalized by hydrogel crosslinking structure (PEG/CMC) on fluoro-MNPs surface by using LbL assembly. These hydrogel properties are used as preserver for protein delivery [3]. ALK1fc as specific TGFβ1 inhibitor is encapsulated inside (PEG/CMC) layers during Layer by Layer (LbL) assembly. Zeta potential measurements, X-rays diffraction and SDS page-silver staining results confirm encapsulation of ALK1fc. Efficiency of encapsulated ALK1fc was measured by Immune-fluorescence assay against localization of TGFβ1. Stained TGFβ1 appeared as purple color, distributed in cytoplasm of control HLF whereas it disappeared in HLF sample treated by encapsulated ALK1fc.
[1] Terreno E, et al., Chem. Rev., 2010, 110, 3019–3042.
[2] Mohapatra,J., et al.,. Nanoscale, 2015, 7, 9174–9184
[3] Saha N. et al., Journal of Biomaterials and Nanobiotechnology, 2 (2011), pp. 85–90.
Acknowledgment: This work was partially supported by PON02_00563_3448479 RINOVATIS. SL also gratefully acknowledges the financial suppport of the REA research grant n° PITN-GA-2012-316549 (IT LIVER) from the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007-2013).
